Various means of detecting chemiluminescence are known and in commercial use. One of the most convenient means is a charge-coupled device (CCD) which is commonly incorporated into a camera (CCD camera). This type of device is rapidly becoming accepted in laboratories because it allows quantitative imaging of virtually any shape object or set of objects and because of the ease of computerized data storage and processing. A unique feature of CCDs is their superior sensitivity to red light. Unfortunately, chemiluminescent compounds which are currently available for qualitative and quantitative detection of enzymes are blue or green emitting. Detection sensitivity of CCDs is markedly inferior at these wavelengths. Accordingly, chemiluminescent compounds which produce light in the red region of the spectrum are required to take full advantage of CCD detection technology.
Alkaline phosphatase (AP) is frequently used as a marker or label in enzyme-linked assays for biological molecules and other analytes of interest such as drugs, hormones, steroids and cancer markers. Chemiluminescent detection of this enzyme offers a safe, convenient and sensitive means to provide a quantitative measure of the amount of enzyme in a sample or of the amount of an enzyme-labeled analyte or labeled specific binding partner for an analyte. No chemiluminescent enzyme substrate in commercial use generates red chemiluminescence. Substrates which are capable of producing red chemiluminescence would prove advantageous when used in conjunction with CCD detection. Such substrates would preferably produce red chemiluminescence with high efficiency and of an extended duration. Both of these goals are met by the compounds and compositions of the present invention.
Applicant's published PCT application WO97/26245 discloses chemiluminescent heterocyclic compounds which produce light upon reaction with a phosphatase. Possible heterocyclic ring fragments include a 2-(4-hydroxy-2-benzothiazolyl)-2-thiazolyl group (luciferyl group).
The light-producing compound occurring in various species of beetles, termed luciferin, are oxidized by a luciferase to produce bioluminescence in vivo ranging from green to orange. Red luminescence can be produced using the native luciferin and luciferase in vitro at pH&lt;7 and by autoxidation of luciferin in DMSO or aqueous base (W. D. McElroy, H. H. Seliger, E. H. White, Photochem. Photobiol., 10, 153-170 (1969)). Synthetic analogs of luciferin such as 4,6-dihydroxyluciferin and 6-aminoluciferin have been reported to generate red bioluminescence on reaction with luciferase (E. H. White, H. Worther, J. Org. Chem. 31, 1484-1488 (1966); E. H. White, H. Worther, H. H. Seliger, W. D. McElroy, J. Am. Chem. Soc., 88, 2015-2019 (1966)). Another analog, 5,5-dimethylluciferin produces red chemiluminescence in oxygenated DMSO or in aqueous alkaline solution but does not produce bioluminescence with luciferase (T. A. Hopkins, H. H. Seliger, E. H. White, M. W. Cass, J. Am. Chem. Soc., 89, 7148 (1967)). It is important to note that none of the red-emitting bio- or chemiluminescent reactions indicated above have found commercial utility in enzyme-linked assays such as immunoassays and DNA probe assays.